Conformational dynamics of the ClpA Hexamer. (GP, AG, MRM)[unreadable] [unreadable] ClpA, a Hsp100/Clp chaperone from E. Coli, is a hexameric ring of subunits that unfolds native proteins in an ATP-dependent process and delivers the unfolded polypeptides to ClpP protease for degradation. Substrate recognition by ClpA is dependent on interactions with specific tags, such as the 11-amino acid SsrA sequence appended to the C-termini of proteins translationally stalled at ribosomes. To gain insight into the overall conformational changes of the ClpA hexamer upon peptide binding, we have used sedimentation velocity measurements to determine sedimentation coefficient (S) distributions of ClpA in the absence and presence of 10-fold excess of SsrA at different temperatures. Previous calorimetric titrations of ClpA hexamer with SsrA have suggested that peptide binding may trigger conformational changes within the ClpA hexamer. Analytical ultracentrifugation studies indicate that peptide-bound ClpA exhibits a strikingly more narrow distribution of sedimentation coefficients than does ClpA sedimenting in the absence of the peptide. Similar results are obtained for the wild-type ClpA and for a deletion mutant lacking the N-terminal regulatory domain (ClpADN). The conformational change in ClpA hexamer induced by SsrA peptide binding, as reflected by the S distribution, is temperature dependent. Overall, sedimentation velocity results indicate that substrate peptide binding stabilizes a unique conformation of the ClpA hexamer.[unreadable] [unreadable] To further study conformational dynamics of the ClpA hexamer and the dynamics of peptide substrate translocation, we are preparing a double cysteine mutant of ClpA. This mutant will be labeled with a donor-acceptor dye pair containing a long fluorescence lifetime donor. Preliminary studies confirmed that energy transfer measurements with metal-ligand complex based fluorophores as donors may reveal information on microsecond dynamics of CpA protein.[unreadable] [unreadable] [unreadable] Oligomerization and proteolytic activity of the ClpP protease from Bacillus subtilis. (GP, AG, MRM, DS)[unreadable] [unreadable] The proteolytic component of the ClpXP and ClpAP complexes, the ClpP protease, is well conserved among different species from eubacteria to the mitochondria of higher eukaryotes. This system has been well studied in the gram-negative bacteria E. Coli, but there is a growing interest in studying ClpP in gram positive bacteria such as Bacillus subtilis. Recently, it has been shown that ClpP is a target of a new class of antibiotic in gram positive bacteria. There is also some evidence suggesting that ClpP may be present in its monomeric form in Bacillus subtilis. We cloned both wilde type and histidine-tagged Bacillus subtilis ClpP gene, expressed this gene in the E. Coli, and purified the proteins. Our data show that cloned Bacillus subtilis ClpP forms functional complexes with E. Coli ClpX proteins. Gel filtration experiments suggest that Bacillus subtilis ClpP forms tetradecamer complexes in vitro. Analytical ultracentrifugation studies of the Bacillus subtilis ClpP are underway.[unreadable] [unreadable] [unreadable] Oxidation dependent oligomerization of the human Peroxiredoxin I (hPrxI) and its interactions with human Sulfiredoxin (hSrx). (GP, AG, DYL, SGR)[unreadable] [unreadable] Human Peroxiredoxin I belongs to a large and diverse family of antioxidant enzymes that also regulate cell signaling pathways, apoptosis and differentiation. Peroxiredoxins (Prxs) can be regulated by changes to phosphorylation and redox state. Oligomerization is believed to play a key role in this regulation. Most of 2-Cys Prxs demonstrate dramatic changes in oligomeric state, forming either dimers or decamers with the characteristic doughnut shape.[unreadable] Currently we are studying redox-dependent oligomerization of hPrx I for both reduced, oxidized and overoxidized (Cys-SO2H) forms of this enzyme and interactions of those complexes with the human Sulfiredoxin (hSrx). Sedimentation velocity experiment show, that hPrx redox-dependent oligomerization is strikingly different from other Prxs, reported in literature. The oxidized hPrx is an obligate homodimer and the reduced form of this enzyme is in concentration dependent monomer-dimer-decamer equilibrium. The overoxidized form of hPrx also forms decamers, and the concentration dependence of this process in higher than that of the reduced form of this enzyme. Multisignal sedimentation velocity method will be used to obtain information on the stoichiometry for interactions of hSrx with different oligomeric forms of hPrx in solution.[unreadable] [unreadable] [unreadable] Capping Protein - CARMIL Interaction Studies. (GP, AG, TU, JAH)[unreadable] [unreadable] Capping Protein (CP) is a highly conserved actin-binding protein that is essential for normal actin dynamics. CP binds to the barbed end of the actin filament blocking both association and dissociation of actin monomers. One potential regulator of CP is CARMIL, that might act as potent CP antagonist and inhibit CP interaction with actin filaments.[unreadable] Interactions of Mouse Capping Protein (mCP) heterodimer (a, b) with different fragments of mouse CARMIL are being studied by isothermal titration calorimetry to identify binding sites. By conducting these experiments at different temperatures, heat capacity information will be obtained.